This application claims benefits of the International Application PCT/JP00/08321 filed Nov. 24, 2000, and the Japanese Patent Application 11345541 filed Dec. 3, 1999.
The present invention relates to a novel polypeptide, a gene encoding the polypeptide, an expression vector for expressing the polypeptide, a transformant obtained by transforming a host cell using the expression vector, and a method for producing a compound useful as a material for synthesis of medicaments and pesticides using the transformant. More particularly, the present invention relates to a polypeptide isolated from a microorganism having an enzyme activity to asymmetrically reduce tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate to tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate, a polynucleotide encoding the polypeptide, an expression vector including the polynucleotide, and a transformant transformed by the expression vector.
The present invention also relates to a method for producing tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate. The method includes the steps of reacting the transformant or a processed product thereof with tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate, and collecting the produced tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate.
Tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate is a compound useful as a material for synthesis of medicaments and pesticides, particularly HMG-CoA reductase inhibitors.
The only method for producing tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate by asymmetrically reducing tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate, which has been reported, employs diethylmethoxyborane and sodium borohydride (U.S. Pat. No. 5,278,313). Problems with this technique are that a very low temperature reaction vessel achieving temperatures as low as xe2x88x9278xc2x0 C. is required, that expensive materials need to be employed, and the like. There has been a demand for a practical reaction procedure.
The inventors of the present invention found a polypeptide derived from a microorganism which asymmetrically reduces tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate to produce tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate, envisioned a method which can efficiently produce tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate, and eventually achieved the present invention.
An objective of the present invention is to provide a polypeptide capable of asymmetrically reducing tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate to produce tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate. Another objective of the present invention is to provide a method for efficiently producing the polypeptide using recombinant DNA technology. Still another objective of the present invention is to provide a transformant which can produce said polypeptide, and a polypeptide having a glucose dehydrogenase activity, simultaneously to a large extent. Even still another objective of the present invention is to provide a practical method for producing tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate using the transformant.
The present invention relates to a polypeptide having an enzyme activity to asymmetrically reduce tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate to tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate. The polypeptide comprises the amino acid sequence represented by SEQ ID NO. 1 in the Sequence Listing, or the amino acid sequence having at least one amino acid substitution, insertion, deletion, or addition thereof.
Preferably, the peptide may be derived from a microorganism belonging to genus Candida. More preferably, the microorganism may be Candida magnoliae IFO 0705.
In one aspect, the present invention relates to a polynucleotide encoding the above-described polypeptide.
In one aspect, the present invention relates to a polynucleotide encoding a polypeptide having an enzyme activity to asymmetrically reduce tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate to tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate. The polynucleotide is hybridized with a nucleotide sequence represented by SEQ ID NO. 2 in the Sequence Listing under stringent conditions.
In one aspect, the present invention relates to an expression vector including the above-described polynucleotide. Preferably, the expression vector may be plasmid pNTCR.
In one embodiment, the above-described expression vector may further include a polynucleotide encoding a polypeptide having a glucose dehydrogenase activity.
Preferably, the polypeptide having a glucose dehydrogenase activity may be a glucose dehydrogenase derived from Bacillus megaterium. 
Preferably, the expression vector may be plasmid pNTCRG.
In one aspect, the present invention relates to a transformant obtained by transforming a host cell using the above-described expression vector.
Preferably, the host cell may be Escherichia coli. More preferably, the transformant may be E. coli HB101 (pNTCR) or E. coli HB101 (pNTCRG).
In one aspect, the present invention relates to a method for producing tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate. The method comprises the steps of reacting the above-described transformant and/or a processed product thereof with tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate, and collecting produced tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate.
Preferably, the reacting step is carried out in the presence of a coenzyme restoring system.